Abstract The Pseudomonas aeruginosa type III secretion system (T3SS) functions like a molecular syringe to inject toxins into host cells. The primary cellular targets are phagocytes and epithelial cells. The injected toxins promote phagocytic avoidance and tissue destruction resulting in systemic spread of the bacteria and overwhelming sepsis. Deployment of the T3SS is highly regulated and only activated when bacteria contact host cell surfaces. The overriding hypothesis guiding the studies is that increased understanding of mechanisms that regulate T3SS gene expression will lead to the identification of critical steps that can be targeted by anti-virulence therapeutics, the long term goal of the proposed work. The primary protein required for contact- dependent activation of the T3SS is a transcription factor called ExsA. Preliminary data demonstrate that exsA transcription, ExsA synthesis, and ExsA activity are each subject to control by global regulatory systems. The goals of this proposal are to define novel mechanisms that control exsA transcription, and possibly ExsA synthesis or activity. Our preliminary data demonstrate that the 300 bp intergenic region located between exsB and exsA functions in two capacities. First, the intergenic region contains a Vfr-dependent promoter dedicated to exsA transcription. Vfr is cAMP-dependent transcription factor previously known to regulate T3SS gene expression but the mechanism has not been defined. Second, expression of the intergenic region in trans has a stimulatory effect on T3SS gene expression. Unclear is whether the stimulatory activity is an RNA or a protein. The three aims will characterize the stimulatory activity of the intergeneic region and factors that regulate intergenic promoter activity as follows: Aim 1. Characterize the stimulatory (trans-acting) activity of the exsB-exsA intergenic region. Aim 2. Characterize IR promoter activity. Aim 3. Determine whether Vfr, MvaT/MvaU, and VqsM function together, antagonistically, or independently to modulate IR promoter activity.